The green alga Planktochlorella nurekis is considered a producer of antibacterial mixture of long-chain fatty acids, which has possibly similar composition and mode of action as chlorellin produced by another green alga, Chlorella vulgaris. Although the antibacterial properties of C. vulgaris have been reported, the interactions of P. nurekis with bacteria have not been determined yet. The aim of this study was to elucidate the effect of P. nurekis water suspension on growth of selected gastrointestinal bacteria in vitro so that it could be used as a suitable feed supplement in animal farming. Unknown bacterial populations occurring in the algal suspension were identified using 16S rRNA sequencing assay. Selected strains were cultivated with lyophilized P. nurekis and the antibacterial effect was monitored. The composition of fatty acids and heat sensitivity of antibacterial substances were also examined. Sequencing analysis of 71 bacterial 16S rRNA genes in xenic algal suspension identified common environmental microbiota, one strain belonging to the class Alphaproteobacteria, 17 to Betaproteobacteria, 44 to Gammaproteobacteria (dominated by Pseudomonas putida strains), and nine to Sphingobacteria. The antimicrobial activity of P. nurekis suspension was tested at a concentration range of 0.75–6 mg/ml. The highest inhibitory effect was observed on bifidobacteria. Statistically significant reductions in bacterial counts were also observed for Escherichia coli, Salmonella enterica var. Enteritidis, S. enterica var. Infantis, Campylobacter jejuni, and Arcobacter butzleri. The growth of Lactobacillus johnsonii was significantly stimulated. The antibacterial compounds present in P. nurekis suspension exhibited thermostability. The results indicate that P. nurekis can inhibit some pathogenic gastrointestinal bacteria and seems to be a promising essential nutrients source in animal nutrition.